肺上皮细胞自噬体在结核分枝杆菌感染中保护作用的分子机制

目的：构建Atg5．真核表达载体并瞬时转染肺上皮细胞细胞株,探讨自噬在结核分枝杆菌感染上皮细胞中保护作用的分子机制。方法：设计针对Atg5的RNAi序列,化学合成后经过变性,退火连接到pSilencerTM3．1-H1hygro真核表达载体,经测序验证其正确性。脂质体法瞬时转染真核细胞A549,免疫印迹法检测瞬时转染的效果。用结核分枝杆菌分别感染正常和自噬表达低下的A549细胞．通过检测LDH来观察细胞的坏死情况。结果：成功的构建了pSilencerTM3．1-H1hygro真核表达载体并建瞬时转染了A549细胞株,成功抑制了细胞的自噬功能。Atg5-细胞对结核杆菌的抵抗能力下降。结论：在自噬表达低下的细胞中,细胞对结核分枝杆菌的抵抗能力有明显下降。在结核分枝杆菌感染上皮细胞的过程中,自噬是一种保护机制。     

Evidence for a small anion-selective channel in the cell wall of Mycobacterium bovis BCG besides a wide cation-selective pore.

Two channels were observed in extracts of whole Mycobacterium bovis BCG cells using organic solvents and detergents. The channels derived from organic solvent treatment had a single-channel conductance of about 4.0 nS in 1 M KCl in lipid bilayer membranes with properties similar to those of the channels discovered previously in Mycobacterium smegmatis and Mycobacterium chelonae. The channel was in its open configuration only at low transmembrane potentials. At higher voltages it switched to closed states that were almost impermeable for ions. Lipid bilayer experiments in the presence of detergent extracts of whole cells revealed another channel with a single-channel conductance of only 780 pS in 1 M KCl. Our results indicate that the mycolic acid layer of M. bovis BCG contains two channels, one is cation-selective and its permeability properties can be finely controlled by cell wall asymmetry or potentials. The other one is anion-selective, has a rather small single-channel conductance and is voltage-insensitive. The concentration of channel-forming proteins in the cell wall seems to be small, which is in agreement with the low cell wall permeability for hydrophilic solutes.     

A characteristic phenolic glycolipid antigen from Mycobacterium haemophilum

A characteristic phenolic glycolipid was detected, by thin layer chromatography, in non-polar lipid extracts of nine representative strains of Mycobacterium haemophilum . The lipid was a specific antigen that reacted strongly with serum raised against whole cells of M. haemophilum. Sera from six other mycobacterial strains were tested but only that from Mycobacterium kansasii give a weak reaction.     

Target cell lysis and IL-2 secretion by gamma/delta T lymphocytes after activation with bacteria

Peripheral blood T lymphocytes from healthy donors were stimulated with Mycobacterium tuberculosis in vitro and afterward analyzed phenotypically. Marked expansion of the gamma/delta T cell population (3- to 21-fold) was observed in 15/21 donors 7 to 10 days after stimulation. In addition to M. tuberculosis, Mycobacterium leprae (six of eight) as well as the gram-positive bacteria, Staphylococcus aureus (two of six), group A streptococci (seven of nine), and Listeria monocytogenes (four of eight) augmented gamma/delta TCR expression in peripheral blood T cells of many donors. gamma/delta T lymphocytes expressed IL-2R and secreted IL-2 upon restimulation with M. tuberculosis. Stimulation with M. tuberculosis evoked specific cytolytic activities in gamma/delta T lymphocytes because: gamma/delta T cells lysed M. tuberculosis pulsed but not unpulsed targets; high concentrations of TCR delta 1 mAb facilitated killing of unpulsed target cells; and low doses of anti-TCR delta 1 mAb blocked killing of pulsed targets. Furthermore, gamma/delta T cells from four donors, after activation with M. tuberculosis or with group A streptococci, respectively, only lysed targets pulsed with the homologous agents, whereas in other donors some cross-reactivity was observed. We conclude that, upon contact with mycobacteria and perhaps other microorganisms, gamma/delta T cells are activated which contribute to immunity against infection via IL-2 secretion and specific target cell lysis.     

Breaking down the wall: fractionation of mycobacteria

Mycobacterium spp. possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan-arabinogalactan complex which in turn is esterified by mycolic acids that form with other non-bound lipids an asymmetric permeability barrier and an outer layer, also called a capsule in the case of pathogenic species. In order to investigate the functional roles of the cell envelope components, especially those of the major pathogens Mycobacterium tuberculosis and Mycobacterium leprae, it is necessary to fractionate the envelope by breaking the unusual wall that covers these bacteria. To this aim we first compared the efficiency of high pressure (cell disrupter/French press) with those of pathogen-compatible breakage methods such as sonication, bead beater and lysozyme treatment using the non-pathogenic Mycobacterium smegmatis. When the distribution of various specific markers of the cell envelope compartments, which include mycolic acids, arabinose, NADH oxidase activity, cell wall and cytosolic proteins, were determined sonication combined with lysozyme treatment was found to be the best option. The protocol of subcellular fractionation was then validated for pathogenic species by applying the method to Mycobacterium bovis BCG cells, an attenuated strain of the M. tuberculosis complex.     

Studies on neutral exopolysaccharides produced by the ectomycorrhiza Thelephora terrestris

We developed an in vitro tissue-culture model to analyze the process involved in mycobacterial spread through lung epithelial cell monolayers. A549 cells were infected with low numbers of viable Mycobacterium tuberculosis bacilli expressing the gfp gene. Subsequent addition of a soft agarose overlay prevented the dispersal of the bacilli from the initial points of attachment. By fluorescence microscopy the bacteria were observed to infect and grow within the primary target cells; this was followed by lysis of the infected cells and subsequent infection of adjacent cells. This process repeated itself until an area of clearing (plaque formation) was observed. The addition of amikacin after initial infection did not prevent intracellular growth; however, subsequent plaque formation was not observed. Plaque formation was also observed after infection with Mycobacterium bovis BCG bacilli, but the plaques were smaller than those formed after infection with M. tuberculosis. These observations reinforce the possibility that cell-to-cell spreading of M. tuberculosis bacilli, particularly early in the course of infection within lung macrophages, pneumocytes, and other cells, may be an important component in the infectious process.     

Further studies of the use of cycloheximide for cultivating Mycobacterium lepraemurium in cell culture

The advantage of using cycloheximide for cultivating Mycobacterium lepraemurium in cell culture was further demonstrated. Continuous multiplication of the bacillus in successive subcultures was obtained in MFP, HEp-2 and Vero cells maintained in culture medium containing 0.1 microgram of cycloheximide per ml. Growth characteristics were comparable to those observed in the cultures of A31 cells previously reported. The procedure was simple and convenient. Comparable results, however, have not been obtained in cultures of other established cell lines, HeLa 229, L, MDCK, and Neuro-2a.     

Uptake of radioactive DOPA by Mycobacterium leprae in vitro.

Our previous studies demonstrated that Mycobacterium leprae contains a characteristic o-diphenoloxidase which converts a variety of phenolic compounds to quinones in vitro. This enzyme was not present in any other mycobacteria tested. The results reported here deal with the uptake and binding of radioactive DOPA by M. leprae. The leprosy bacilli incubated with tritium-labelled DOPA, readily took up the substrate. The binding of DOPA by the bacilli was markedly inhibited by diethyldithiocarbamate. The organisms also bound tritiated norepinephrine. Mycobacterium phlei which does not oxidize phenolic substrates failed to bind DOPA. Cultures of melanocytes which contain o-diphenoloxidase took up tritiated DOPA. Catecholamine metabolism is known to be important in myocardial cells. Cultures of turtle-heart cells did not oxidize DOPA to quinone; however, these cells bound the labelled substrate. A cell line of fibroblasts derived from armadillo skin neither oxidized nor took up DOPA. The results indicate that, like melanocytes and turtle-heart cells, M. leprae probably possesses specific receptor sites for the binding and subsequent metabolism of phenolic substrates.     

Analysis of mitogen-stimulated lymphocyte subset proliferation and nitric oxide production by peripheral blood mononuclear cells of captive elk (Cervus elaphus)

Elk (Cervus claphus) are reservoirs for Brucella abortus, Mycobacterium bovis, and Mycobacterium avium subsp. paratuberculosis, each a serious pathogen of domestic livestock. An understanding of the basic immune responsiveness of elk would aid efforts to develop methods to diagnose and prevent these diseases of elk. Peripheral blood mononuclear cells (PBMC) isolated from captive elk were examined for phenotype, lymphocyte subset proliferative capacity, and ability to produce nitric oxide (NO) upon pokeweed mitogen (PWM) stimulation. Although gamma delta TCR+ cells represented a high percentage of the peripheral blood lymphocyte pool, these cells responded poorly to PWM stimulation. B cells (i.e., sIgM+ cells), conversely, were responsive to PWM stimulation. Addition of PWM to PBMC cultures also resulted in a significant production of nitrite, the stable oxidation product of NO. Similar to other ruminant species, the majority of elk peripheral blood sIgM+ cells co-expressed MHC class II and B-B4, a B cell lineage marker that varies in expression during B cell development. Findings from the present study provide basic information on several parameters of cellular immunity of elk.     

Truncated structural variants of lipoarabinomannan in Mycobacterium leprae and an ethambutol-resistant strain of Mycobacterium tuberculosis

Current knowledge on the structure of lipoarabinomannan (LAM) has resulted primarily from detailed studies on a few selected laboratory strains of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium smegmatis. Our previous work was the first to report on the salient structural features of M. tuberculosis clinical isolates and demonstrated significant structural variations. A prime effort is to correlate a particular structural characteristic with observed differences in eliciting an immunobiological response, especially in the context of CD1-restricted presentation of LAM to T cells. T cell clones derived from the cutaneous lesions of leprosy patients have been shown to recognize specifically LAM from Mycobacterium leprae and not from M. tuberculosis Erdman or H37Rv. Herein we provide further fine structural data on LAM from M. leprae (LepLAM) and a tuberculosis clinical isolate, CSU20 (CSU20LAM), which was unexpectedly recognized by the supposedly LepLAM-specific CD1-restricted T cell clones. In comparison with the de facto laboratory LAM standard from M. tuberculosis H37Rv (RvLAM), LepLAM derived from in vivo grown M. leprae is apparently simpler in its arabinan architecture with a high degree of exposed, non-mannose-capped termini. On the other hand, CSU20, an ethambutol-resistant clinical isolate, makes a vastly heterogeneous population of LAM ranging from rather small and non-mannose-capped to full-length and fully capped variants. LepLAM and CSU20LAM contain a higher level of succinylation than RvLAM, which, in the context of truncated or less elaborated arabinan, may contribute to selective recognition by T cells. LAM from all species could be resolved into discrete forms by isoelectric focusing based apparently on their arabinan heterogeneity. In the light of our current and more recent findings, we reason that all immunobiological data should be cautiously interpreted and that the actual LAM variants that may be present in vivo during infection and pathogenesis need to be taken into consideration.     

Mycobacterium leprae inhibits dendritic cell activation and maturation

Leprosy presents with a clinical spectrum of skin lesions that span from strong Th1-mediated cellular immunity and control of bacillary growth at one pole to poor Ag-specific T cell immunity with extensive bacillary load and Th2 cytokine-expressing lesions at the other. To understand how the immune response to Mycobacterium leprae is regulated, human dendritic cells (DC), potent inducers of adaptive immune responses, exposed to M. leprae, Mycobacterium tuberculosis (Mtb), and Mycobacterium bovis bacillus Calmette-Guerin (BCG) were studied for their ability to be activated and to prime T cell proliferation. In contrast with Mtb and BCG, M. leprae did not induce DC activation/maturation as measured by the expression of selected surface markers and proinflammatory cytokine production. In MLR, T cells did not proliferate in response to M. leprae-stimulated DC. Interestingly, M. leprae-exposed MLR cells secreted increased Th2 cytokines as well as similar Th1 cytokine levels as compared with Mtb- and BCG-exposed cells. Gene expression analysis revealed a reduction in levels of mRNA of DC activation and maturation markers following exposure to M. leprae. Our data suggest that M. leprae does not induce and probably suppresses in vitro DC maturation/activation, whereas Mtb and BCG are stimulatory.     

Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines

The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT-PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25-kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20-amino-acid-long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross-linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins.     

TNF-α-mediated activation of HIV-1 LTR in monocytoid cells by mycobacteria

Mycobacterial infection occurs commonly in patients with acquired immune deficiency syndrome. Incubation of monocytoid cell line U937 cells, which was cotransfected HIV-1 long terminal repeat sequence (LTR) chloramphenicol acetyltransferase (CAT) plasmid and Tat expression plasmid, with Mycobacterium smegmatis, Mycobacterium avium, Mycobacterium bovis BCG and Mycobacterium tuberculosis resulted in enhancement of CAT production, indicating that these mycobacteria could activate LTR in this cell line. The amount of CAT in the cells coexisting with M. smegmatis was higher than that infected with other mycobacteria. The amounts of CAT production in the cells coculturing with M. avium and M. bovis BCG were intermediate. M. tuberculosis slightly stimulated CAT production. The amount of tumor necrosis factor (TNF)-alpha produced by transfected U937 cells was correlated with the amount of CAT production. The interleukin (IL)-1beta and IL-6 levels in the supernatant from coculturing with all species were similar. The antibody to TNF-alpha inhibited CAT production induced by mycobacterial infections. The anti-IL-1beta and anti-IL-6 antibodies, however, scarcely influenced stimulation of LTR by mycobacteria. In addition, U937 cells transfected with full length LTR CAT plasmid showed increased CAT production by activation with mycobacteria, but the cells transfected with mutant LTR CAT constructs from which the nuclear factor (NF)-kappaB binding site was deleted did not show activation. These findings indicated that activation of Mycobacterium-induced LTR CAT is NF-kappaB dependent. These findings suggested that activation of HIV-1 LTR by mycobacteria was mainly mediated by NF-kappaB-induced secondary release of cytokine TNF-alpha.     

Mycobacterial proteome extraction: comparison of disruption methods

Mycobacterium avium subsp. paratuberculosis has long been recognized as the causative agent of Johne's disease, a chronic inflammatory intestinal disease of sheep, cattle and other ruminants. Mycobacterial cells are extremely hardy, and proteomic analyses require the use of harsh conditions to effect their disruption. We compared the effectiveness of bead beating and sonication as cell lysis methods for the extraction of the proteomes of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis. Broad and narrow range two-dimensional gel electrophoresis was used to compare the numbers of silver stained protein spots that were observed in mycobacterial lysates. Despite differences in the yield of total protein from either species, and at different ages, the two methods appeared to give similar representations of the mycobacterial proteomes analyzed. Bead beating therefore represents a rapid and effective method of extracting the proteomes of mycobacterial species without the risks associated with an open tube sonication procedure.     

Human toll-like receptors mediate cellular activation by Mycobacterium tuberculosis.

Recent studies have implicated a family of mammalian Toll-like receptors (TLR) in the activation of macrophages by Gram-negative and Gram-positive bacterial products. We have previously shown that different TLR proteins mediate cellular activation by the distinct CD14 ligands Gram-negative bacterial LPS and mycobacterial glycolipid lipoarabinomannan (LAM). Here we show that viable Mycobacterium tuberculosis bacilli activated both Chinese hamster ovary cells and murine macrophages that overexpressed either TLR2 or TLR4. This contrasted with Gram-positive bacteria and Mycobacterium avium, which activated cells via TLR2 but not TLR4. Both virulent and attenuated strains of M. tuberculosis could activate the cells in a TLR-dependent manner. Neither membrane-bound nor soluble CD14 was required for bacilli to activate cells in a TLR-dependent manner. We also assessed whether LAM was the mycobacterial cell wall component responsible for TLR-dependent cellular activation by M. tuberculosis. We found that TLR2, but not TLR4, could confer responsiveness to LAM isolated from rapidly growing mycobacteria. In contrast, LAM isolated from M. tuberculosis or Mycobacterium bovis bacillus Calmette-Guérin failed to induce TLR-dependent activation. Lastly, both soluble and cell wall-associated mycobacterial factors were capable of mediating activation via distinct TLR proteins. A soluble heat-stable and protease-resistant factor was found to mediate TLR2-dependent activation, whereas a heat-sensitive cell-associated mycobacterial factor mediated TLR4-dependent activation. Together, our data demonstrate that Toll-like receptors can mediate cellular activation by M. tuberculosis via CD14-independent ligands that are distinct from the mycobacterial cell wall glycolipid LAM.     

Mycobacterium tuberculosis mammalian cell entry operon (mce) homologs in Mycobacterium other than tuberculosis (MOTT)

The cloned mammalian cell entry gene mce1a from Mycobacterium tuberculosis confers to non-pathogenic Escherichia coli the ability to invade and survive inside macrophages and HeLa cells. The aim of this work was to search for and characterize homologs of the four M. tuberculosis mammalian cell entry operons (mce1, mce2, mce3 and mce4) in mycobacteria other than tuberculosis (MOTT). The dot-blot and polymerase chain reaction (PCR) experiments performed on 24 clinical isolates representing 20 different mycobacterial species indicated that the mce operons were widely distributed throughout the genus Mycobacterium. BLAST search results showed the presence of mce1, mce2 and mce4 homologs in Mycobacterium bovis, Mycobacterium avium and Mycobacterium smegmatis. A homologous region for the mce3 operon was also found in M. avium and M. smegmatis. DNA and protein alignments were done to compare the M. tuberculosis mce operons and the deduced M. bovis, M. avium, and M. smegmatis homologs. The deduced proteins of M. bovis mce1, mce2 and mce4 operons had 99.6-100% homology with the respective M. tuberculosis mce proteins (MTmce). The similarity between M. avium mce proteins and the individual M. tuberculosis homologs ranged from 56.2 to 85.5%. The alignment results between M. smegmatis mce proteins and the respective MTmce proteins ranged from 58.5% to 68.5%. Primer sets were designed from the M. tuberculosis mce4a gene for amplification of 379-bp fragments. Amplification was successful in 14 strains representing 11 different mycobacterial species. The PCR fragments were sequenced from 10 strains representing eight species. Alignment of the sequenced PCR products showed that mce4a homologs are highly conserved in the genus Mycobacterium. In conclusions, the four mce operons in different mycobacterial species are generally organized in the same manner. The phylogenetic tree comparing the different mce operons showed that the mce1 operon was closely related to the mce2 operon and mce3 diverged from the other operons. The wide distribution of the mce operons in pathogenic and non-pathogenic mycobacteria implicates that the presence of these putative virulence genes is not an indicator for the pathogenicity of the bacilli. Instead, the pathogenicity of these factors might be determined by their expression.     

Human T cell clones recognize two abundant Mycobacterium tuberculosis protein antigens expressed in Escherichia coli

Human T cells reactive to Mycobacterium tuberculosis were cloned from peripheral blood mononuclear cells (PBMC) of four tuberculosis patients by using whole irradiated bacilli as the in vitro stimulatory agent. Twenty-two T cell clones (CD4+) were tested for their reactivity to 12 different mycobacterial species and showed a distribution from limited to broad cross-reactivity. These T cell clones were also tested for their reactivity to three abundant M. tuberculosis proteins of 65, 19, and 14 kD, each expressed from recombinant DNA in Escherichia coli. The three proteins were expressed from DNA clones that were previously isolated from a lambda gt11 genomic DNA library of M. tuberculosis by using monoclonal antibodies directed against this pathogen. A T cell clone from one patient was stimulated by an E. coli lysate containing the 65 kD antigen, and a T cell clone from a second patient was stimulated by an E. coli lysate containing the 19 kD antigen in an in vitro proliferation assay. Both T cell clones showed very limited cross-reactivity when tested against other mycobacteria. We conclude that some patients with tuberculosis exhibit a T cell response to the 65 and 19 kD M. tuberculosis proteins defined by these isolated genes.     

Formation of nonculturable Mycobacterium tuberculosis and their regeneration

Nonculturable cells were found to occur in populations of Mycobacterium tuberculosis cells during the long post-stationary phase. These cells were small (0.6-0.8 micron) ovoid and coccoid forms with intact cell walls and negligible respiratory activity, which allows them to be regarded as dormant cells. Nonculturable cells were characterized by low viability after plating onto solid medium; a minor part of the population of these cells could be cultivated in liquid medium. Cell-free culture liquid of an exponential-phase Mycobacterium tuberculosis culture or the bacterial growth factor Rpf exerted a resuscitating effect, increasing substantially the growth capacity of the nonculturable cells in liquid medium. During resuscitation of nonculturable cells, a transition from ovoid to rodlike cell shape occurred. At early stages of resuscitation, ovoid cells formed small aggregates. The recovery of culturability was associated with the formation of rod-shaped cells in the culture. The data obtained demonstrate the in vitro formation of dormant cells of Mycobacterium tuberculosis, which do not grow on solid media but can be resuscitated in liquid medium under the effect of substance(s) secreted by actively growing cells.     

A cell-penetrating peptide derived from mammalian cell uptake protein of Mycobacterium tuberculosis

A Mycobacterium tuberculosis membrane protein called Mycobacterium cell entry protein (Mce1A) was previously shown to mediate the uptake of nonpathogenic Escherichia coli and latex beads by nonphagocytic mammalian cells. Here we characterize further the in vitro invasive activity of Mce1A using colloidal gold nanoparticles and fluorescent latex microspheres. Mce1A-coated colloidal gold particles induced plasma membrane invagination and entered membrane-bound compartments inside HeLa cells. Few of the protein-coated particles were also found in the cytosol compartment. Cytochalasin D and nocodazole inhibited the uptake by HeLa cells, indicating that rearrangement of both microtubules and microfilaments was necessary for the uptake. The functional domain of Mce1A for invasion was narrowed to a highly basic 22-amino acid sequence termed Inv3. A synthetic Inv3 peptide stimulated uptake of colloidal gold particles as well as latex microspheres by HeLa cells. A chimeric protein composed of Inv3 sequence at the N terminus of beta-galactosidase appeared to stain the nuclear membrane, suggesting that it entered the HeLa cell cytoplasm. These observations suggest that the cell uptake activity of Mce1A is confined to a small peptide domain located in the core region of the protein. Inv3 could be used to ferry any protein in fusion with it into mammalian cells and may serve as a potent nonviral delivery system.